Accepted Manuscript

Enhanced hydr ogen pr oduction of Enterobacter aerogenes mutated by nu

clear ir r adiation

Jun Cheng, Min Liu, Wenlu Song, Lingkan Ding, Jianzhong Liu, Li Zhang, Kefa
Cen

PII: S0960-8524(16)31701-1
DOI: http://dx.doi.org/10.1016/j.biortech.2016.12.033
Reference: BITE 17409

To appear in: Bioresource Technology

Received Date: 14 October 2016

Revised Date: 8 December 2016
Accepted Date: 9 December 2016

Please cite this article as: Cheng, J., Liu, M., Song, W., Ding, L., Liu, J., Zhang, L., Cen, K., Enhanced hydr ogen
pr oduction of Enterobacter aerogenes mutated by nuclear ir r adiation, Bioresource Technology (2016), doi:
http://dx.doi.org/10.1016/j.biortech.2016.12.033

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1 Enhanced hydrogen production of Enterobacter aerogenes
2 mutated by nuclear irradiation
3 Jun Chenga,∗, Min Liua, Wenlu Songa,b, Lingkan Dinga, Jianzhong Liua, Li Zhangc, Kefa Cena

a
4 State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China

b
5 Department of Life Science and Engineering, Jining University, Jining 273155, China

c
6 Shandong Botong New Energy Company, Jining 272000, China

7 Abstract:

8 Nuclear irradiation was used for the first time to generate efficient mutants of

9 hydrogen-producing bacteria Enterobacter aerogenes, which were screened with larger colour

10 circles of more fermentative acid by-products. E. aerogenes cells were mutated by nuclear

11 irradiation of 60Co γ-rays. The screened E. aerogenes ZJU1 mutant with larger colour circles

12 enhanced the hydrogenase activity from 89.8 of the wild strain to 157.4 mL H2/(g DW· h). The

13 hereditary stability of the E. aerogenes ZJU1 mutant was certified after over ten generations of

14 cultivation. The hydrogen yield of 301 mL H2/g glucose with the mutant was higher by 81.8% than

15 that of 166 mL/g glucose with the wild strain. The peak hydrogen production rate of 27.2 mL/(L· h)

16 with the mutant was higher by 40.9% compared with that of 19.3 mL/(L· h) with the wild strain. The

17 mutant produced more acetate and butyrate but less ethanol compared with the wild strain during

18 hydrogen fermentation.

19 Keywords: Hydrogen, Enterobacter aerogenes, Nuclear irradiation, Bacteria mutant.

∗
Corresponding author: Prof. Dr. Jun Cheng, State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China. Tel.: +86 571

87952889; fax: +86 571 87951616. E-mail: juncheng@zju.edu.cn

1
1 1. Introduction

2 Renewable energy resources have attracted increasing attention because of the consumption of

3 fossil fuels (Lin et al., 2008). From the perspective of economic development and ecological

4 environment protection, hydrogen has been considered one of the most advantageous alternative

5 source of energy because of its clean combustion product, namely, water (Nitsch et al., 1992).

6 Microbial fermentation is a potentially important method for renewable hydrogen production that

7 uses biomass as substrate. Firstly, the method is carbon neutral and requires less energy

8 consumption as compared with other conventional hydrogen-producing methods, such as steam

9 reforming, partial oxidation, gasification and water electrolysis (Jones, 2008; Angenent et al., 2004;

10 Kothari et al., 2008; Holladay et al., 2009). Secondly, dark fermentation with biomass or

11 carbohydrate-based substrates presents a more promising and feasible route for biological hydrogen

12 production, thereby offering higher production efficiency, higher stability, simpler control

13 requirements, lower operating costs and higher feasibility for industrialisation (Jayasinghearachchi

14 et al., 2009). As a facultative anaerobe, Enterobacter aerogenes has high growth and hydrogen

15 production rates, which have been widely studied in dark fermentation (Zhang et al., 2011).

16 Nevertheless, for commercialisation in biological fermentative hydrogen production, its hydrogen

17 yield needs to be further improved. The theoretical hydrogen yield of E. aerogenes is 4 mol/mol

18 glucose (Zhang et al., 2011; Song et al., 2011). However, the hydrogen yield of E. aerogenes is

19 approximately 1 mol/mol of glucose at pH of 6.0, which is considerably lower than the theoretical

20 value (Tanisho, 1999; Yokoi et al. 1995; Tanisho et al., 1989; Converti and Perego, 2002; Zhang et

21 al., 2011). Therefore, extensive research on E. aerogenes has been conducted to improve the

2
1 hydrogen yield; thus far, a yield of 1.58 mol H2/mol glucose has been experimentally reached

2 mainly by optimising the cultivation conditions (Tanisho et al., 1998).

3 Several approaches, such as genetic engineering and metabolic engineering technologies for

4 most promising micro-organisms, were recently conducted to enhance microbial hydrogen

5 productivity (Rachman et al., 1997; Lu et al., 2008; Lu et al., 2011). However, the hydrogen yield

6 of E. aerogenes remains markedly lower than the theoretical hydrogen yield of 4 mol/mol glucose.

7 Complex gene analysis of various bacteria and the specific methods for different bacteria present

8 considerable limitations for further research. Microbial mutation breeding could obtain stable

9 genetic mutant strains with improved target properties; this approach has been widely used in

10 industrial fermentation (Shi and Wu, 2013). The application of mutation techniques to improve the

11 productivity of hydrogen-producing bacteria is a promising research direction. The E. aerogenes

12 AY-2 mutant was obtained by allyl alcohol and proton suicide methods; its hydrogen yield of 1.17

13 mol H2/mol glucose was higher by 109% than that of the wild strain HU-101 (Rachman et al.,

14 1997). Lu et al. (2008) used He–Ne laser irradiation to improve the hydrogenase activity of the E.

15 aerogenes HB-5M mutant, which had a hydrogen yield of 0.95 mol H2/mol glucose; the maximum

16 specific rate of hydrogen production by the mutant was twice that of the wild strain W-23 (Lu et al.,

17 2008). Numerous other positive mutants have been generated with atmospheric and room

18 temperature plasma; the hydrogen yield of these mutants was approximately 1.134 mol/mol

19 glucose, which was an increase of 26.4% (Lu et al., 2011). Evidently, the mutation breeding method

20 can improve the hydrogen production of E. aerogenes. However, the enhancement of hydrogen

21 yield needs further study.

3
1 Mutagenesis by γ-ray irradiation can cause morphological and biochemical changes in cells

2 (Feng et al., 2015). A large number of free radicals generated by γ-rays in the cells attack the key

3 functional genes and cause gene breakage and recombination because γ-rays interact with

4 molecules and atoms, especially with water molecules (Kovacs and Keresztes, 2002). γ-Rays

5 possess better mutagenic ability and strong penetrability compared with other forms of radiation.

6 Cheng et al. (2014) significantly increased the growth rate and lipid productivity of microalgae

7 using γ-rays irradiation. To the best of our knowledge, studies concerning hydrogen-producing

8 bacteria mutated by γ-ray irradiation have yet to be reported. A large number of samples are

9 randomly selected to test the hydrogen yield in a batch experiment after irradiation to screen the

10 positive mutant strains (Ren et al., 2006). It’s quite difficult to test each strain’s hydrogen yield or

11 producing rate to screen the positive mutant strains because of tremendous bacteria strains.

12 Although positive mutant stains could be obtained by large-scale experiments on selection and

13 fermentation, this traditional method is time-consuming, inefficient and difficult to perform.

14 To our best knowledge, no studies on E. aerogenes mutated by 60Co γ-rays to enhance

15 hydrogen production have been reported in literature to date. γ-rays possess better mutagenic ability

16 and strong penetrability compared with other forms of radiation. The mutation process of γ-ray

17 irradiation has the advantages of high efficiency and low cost. The screened E. aerogenes mutant

18 with high hydrogen productivity has the potential to be utilized on the biological hydrogen

19 production through the anaerobic fermentation of biomass wastes, such as molasses wastewater and

20 food waste in future industrial applications. This paper is intended to fill in this gap in the state of

21 the art. Thus, the objectives of the present study are to:

4
1 (1) Screen E. aerogenes positive mutants with improved hydrogen production using colour

2 circles of acid by-products.

3 (2) Improve hydrogenase activity and hydrogen production of E. aerogenes, and compare

4 metabolic by-products of the mutant and wild strains.

5 (3) Examine the hereditary stability of the screened E. aerogenes ZJU1 mutant.

6 2. Materials and Methods

7 2.1. Bacterial strains and medium

8 Enterobacter aerogenes ATCC13408 was purchased from China General Microbiological

9 Culture Collection Centre. The Luria Bertani (LB) culture medium contained 5 g/L yeast extract, 10

10 g/L peptone and 10 g/L NaCl. The solid LB medium also contained 20 g/L agar.

11 2.2. Mutagenesis and selection of E. aerogenes

12 1 mL of the overnight seed culture of E. aerogenes ATCC13408 was inoculated into 100 mL

13 of the LB medium. Batch cultivations were performed aerobically in a shaker incubator at 37 °C

14 and 180 rpm for 6–8 h until the cells reached the mid-log phase. The bacterial cell culture was

15 centrifuged to prepare a bacterial suspension with normal saline. Six centrifuge tubes (5 mL) with 1

16 mL of bacterial suspension were immersed in ice water and irradiated with 0, 200, 400, 600, 800

17 and 1000 Gy of 60Co γ-rays, respectively (Zhejiang Institute of Agriculture and Nuclear Application

18 Technology, Zhejiang, China). All samples were immersed in ice water for 1–2 h after irradiation to

19 inhibit the activity of intracellular repair enzymes.

20 A 0.1 mL aliquot of each sample was diluted with equal amounts of LB medium. The diluted

21 samples were evenly applied on the solid medium in an incubator (37 °C) for 24 h. The lethality
5
1 percentage of E. aerogenes irradiated by different doses of 60Co γ-rays was calculated based on the

2 following equation:

3 Lethality percentage = [(A − I)/A] × 100% , (1)

4 where A is the total colony count of the sample without irradiation and I is the total colony count

5 irradiated by 60Co γ-rays. All colony numbers were obtained by the CFU (colony-forming unit)

6 method on solid medium.

7 The irradiated samples of each bacterial suspension were respectively diluted and evenly

8 applied on the screening medium. The screening medium contained 20 g/L of glucose, 5 g/L of

9 yeast extract, 10 g/L of peptone, 10 g/L of NaCl, 20 g/L of agar and 0.4 g/L of bromcresol purple.

10 Yeast, peptone and NaCl were same with the culture medium to help bacterial cells grow and

11 reproduce. The screening medium was used for selecting positive mutants by hydrogen production.

12 Glucose substrate was essential to provide suitable carbon source for hydrogen and metabolite

13 production in dark fermentation. The colour of bromcresol purple will change from purple to yellow

14 in acid condition. The size of the yellow circle visually shows the quantity of acid by fermentation,

15 and then indirectly evaluates hydrogen productivity of strains. If one mutant is surrounded by larger

16 yellow circle, it has a larger yield of acid by-products. It’s probable that the yield of acetic acid or

17 butyric acid is positively correlated with the hydrogen yield of the mutant. Positive mutants can be

18 screened based on sizes of yellow circles by the pH indicator. Several well-grown strains with

19 larger yellow circles were selected from the screening medium in an incubator (37 °C) after 12–24

20 h. Sole cells of the mutant strains were selected with an inoculation loop and used to inoculate 100

21 mL LB medium; these cells were cultured until the mid-log phase. The hydrogen yields of the

6
1 selected mutants with larger yellow circles were verified by fermentative experiments to further

2 screen the mutant with markedly improved hydrogen productivity. Eventually, a mutant E.

3 aerogenes ZJU1 with a significantly increased hydrogen yield was obtained.

4 2.3. Hydrogen production by fermentation

5 1 mL each of the overnight seed cultures of E. aerogenes ATCC13408 and E. aerogenes ZJU1

6 were respectively inoculated into 100 mL of LB medium. Batch cultivations were performed for 6 h

7 at 37 °C and 180 rpm aerobically in an incubator shaker until the cells reached mid-log phase.

8 Bacterial cells were added to 200 mL of the fermentative medium, which contained 3 g glucose and

9 0.03 g FeCl2, with 1 mL vitamin liquid and 1 mL microelement liquid. The vitamin liquid contained

10 0.01 g/L glutamic acid, 0.025 g/L ascorbic acid, 0.025 g/L riboflavin, 0.02 g/L citric acid

11 monohydrate, 0.01 g/L folic acid, 0.01 g/L p-aminobenzoic acid and 0.025 g/L creatide. The

12 microelement liquid contained 0.01 g/L MnCl2, 0.05 g/L ZnCl2, 0.01 g/L H3BO3, 0.01 g/L CaCl2,

13 0.01 g/L Na2MoO4, 0.2 g/L CoCl2·6H2O, 0.01 g/L AlK(SO4)2 and 0.01 g/L NiCl2·6H2O. Batch

14 hydrogen production experiments were performed in 350 mL glass bottles in triplicate. Each bottle

15 was purged with N2 gas for 10 min to ensure an anaerobic environment. The pH value was adjusted

16 to 6.0 ± 0.2 by adding 6 M NaOH every 12 h during hydrogen fermentation. The fermentation

17 temperature was maintained at 37 °C in a thermostatic water bath.

18 2.4. Hydrogenase assay

19 1 mL of the fermentative liquid with actively-fermenting cells was collected from the

20 wild-type E. aerogenes and E. aerogenes ZJU1 suspensions to quantify the hydrogenase activity.

21 The culture was moved into a sealed 13 mL vial, which was purged with N2 gas in advance. The

7
1 cells were lysed with glass beads in 2 mL of the anaerobic induction buffer and 200 µl of the

2 sodium–dithionite solution (Song et al., 2011). The vial was purged with N2 gas to ensure an

3 anaerobic environment before 2 mL of Na-dithionite-reduced methyl viologen (MV) buffer was

4 added. The hydrogenase released from the lysed cells was used to produce H2 from the reduced

5 MV. The amount of evolved H2 was used to evaluate the hydrogenase activity of both strains

6 (Carrieri et al., 2008; Zorin, 1986; Peck and Gest, 1956).

7 2.5. Analytical methods

8 The experiments were conducted in triplicates for all the conditions. The results were

9 expressed as mean (± standard deviation). The optical density of wild and mutant E. aerogenes cells

10 at 600 nm (OD600) was measured on a spectrophotometer (Unico UV-2600, China). The biogas

11 produced from batch experiments for hydrogen production was measured every 12 h. The

12 concentrations of H2 and CO2 were analysed by gas chromatography (GC) apparatus (Agilent

13 7820A, USA) equipped with a thermal conductivity detector. The metabolic by-products mainly

14 consisted of ethanol, acetate and butyrate. These by-products were determined by another GC

15 apparatus (Agilent 7820A) equipped with a flame ionisation detector. Both measurements of biogas

16 and metabolic by-products were both previously described in detail (Lin et al., 2015).

17 The dynamic parameters of cumulative hydrogen yield were simulated by the modified

18 Gompertz equation with Origin 8.0.

19 H = Hm exp｛−exp [Rme/Hm(λ − t) + 1]｝, (2)

8
1 where H is the cumulative volume of hydrogen yield (mL), Hm is the maximum hydrogen

2 production potential (mL), Rm is the maximum hydrogen production rate (mL/h), e is 2.718, λ is the

3 lag-phase time (h) and t is the incubation time (h). The overall volumetric hydrogen production rate

4 (Roverall) is defined in the following equation as an index for evaluating the performance of hydrogen

5 production because both the rate and time delay were taken into consideration (Su et al., 2009).

6 Roverall = Hm/｛[(Hm/Rm) + λ]V｝, (3)

7 where V represents the working volume of the culture.

8 3. Results and discussion

9 3.1. Mutation and selection of mutants with improved hydrogen productivity

10 After irradiation with 60Co γ-rays, the E. aerogenes cell suspension was diluted and evenly

11 applied on the solid medium. Bacterial strains were cultivated in an incubator (37 °C) for 24 h. The

12 lethality percentages of E. aerogenes irradiated by different doses of 60Co γ-rays are shown in Fig.

13 1. More positive mutant strains were obtained when the radiation dose was below 400 Gy.

14 However, these positive mutants barely showed any improvements in the hydrogen productivity at

15 lethality percentages of 40%–70%. As the dose was increased, the percentage of bacterial lethality

16 was increased, whereas the positive mutation rate was decreased. More negative mutants but fewer

17 positive mutants were obtained when the dose exceeded 800 Gy (Shi and Wu, 2013). Therefore, the

18 screening positive mutants with improved hydrogen yield is beneficial when the percentage of E.

19 aerogenes lethality is approximately 80%–90%.

20 The lethality rate of E. aerogenes irradiated by 60Co γ-rays was 84% at 600 Gy. As shown in

21 Supplemental Fig. 1, the irradiated cell suspension was diluted and evenly applied on the screening
9
1 medium. Most of the strains were only 3-4 mm, and the yellow circle diameters were 6-8 mm. The

2 well-grown mutant strains (5-6 mm) with larger yellow circles (diameter: 8-12mm) of more

3 fermentative acid by-products were selected. According to the hydrogen-producing equations of E.

4 aerogenes (Eqs. (4) and (5)), acetic acid and butyric acid were also generated during hydrogen

5 production (Hawkes et al., 2007). Theoretically, if the hydrogen yield were improved, the yield of

6 acetic acid or butyric acid would also increase. Mutants with higher hydrogen productivity can be

7 obtained by selecting for those with improved acid productivity based on the sizes of their coloured

8 circles in the culture medium. Fermentative experiments verified the hydrogen productivity of these

9 selected mutants that formed larger yellow circles. Eventually, the E. aerogenes ZJU1 mutant was

10 obtained; the hydrogen yield of this strain was significantly increased.

11 (4)

12 (5)

13 Growth of E. aerogenes ZJU1 mutant and the wild cells was tested. 1 mL culture of the wild

14 and mutant were respectively inoculated into 100 mL of the LB medium and the values of OD600

15 were set as 0 for the very low cell concentration. At first, E. aerogenes ZJU1 grew faster than the

16 wild because of the larger size of mutant cell. After 3 hours, OD600 of the mutant and wild were

17 1.7 and 1.25, respectively, but the growth rate of the wild became faster. After 12 hours, OD600 of

18 the mutant was 3.32, and that of the wild strain was 3.46. Eventually, the value of OD600 with E.

19 aerogenes ZJU1 was slightly lower than that of the wild, and the little difference barely affected the

20 hydrogen yield.

21 3.2. Hydrogenase activity

10
1 To elucidate the mutagenic mechanism of E. aerogenes irradiated by 60Co γ-rays, the

2 hydrogenase activities of the wild and mutant strains were investigated. The hydrogenase activity is

3 defined as the hydrogen yield from 1 g cells in dry weight (DW) per hour. As shown in Fig. 2, the

4 hydrogenase activity of 157.4 ± 8.9 mL H2/(g DW·h) with the E. aerogenes ZJU1 mutant was

5 higher by 75.3% than that of 89.8 ± 7.5 mL H2/(g DW· h) with the wild strain. The hydrogenase

6 activity of E. aerogenes was significantly improved by 60Co γ-ray irradiation. This improvement

7 may be attributed to the 60Co γ-ray irradiation, which causes mutagenesis in the gene encoding

8 enzymes, thereby increasing the active hydrogenase in cells.

9 3.3. Hydrogen productivity

10 To improve the hydrogen yield of both strains, 1% (w/v) malt extract and 0.4% (w/v) yeast

11 extract were added to the initial fermentation medium (containing 1% glucose) for promoting cell

12 growth and reproduction (Song et al., 2011). Fig. 3 shows the results of hydrogen production with

13 both strains during fermentation. As shown in Fig. 3a, the maximum specific rate of hydrogen

14 production with the mutant strain E. aerogenes ZJU1 was 27.19 ± 1.79 mL/(L· h), which was

15 significantly higher than that of the wild strain (19.33 ± 0.81 mL/(L· h)). Moreover, the peak for the

16 mutant strain lasted longer to generate more hydrogen. As shown in Fig. 3b, the cumulative

17 hydrogen yield of 903.09 ± 29.30 mL with the mutant was higher by 81.8% than the yield of 496.74

18 ± 20.65 mL with the wild strain. The hydrogen yield increased from 1.32 ± 0.06 mol/mol glucose to

19 2.41 ± 0.08 mol/mol glucose after E. aerogenes was irradiated by 60Co γ-rays. The dynamic

20 parameters of hydrogen production with the wild and mutant strains are shown in Table 1. The

21 Roverall of E. aerogenes ZJU1 was 15.8 mL/L/h, which increased by 71.7% compared with that of
11
1 the wild type. The lag-phase time for wild E. aerogenes and E. aerogenes ZJU1 were 27.2 and

2 32.7 h, respectively. Hydrogen is mainly produced by Enterobacter species via one of the following

3 methods. First, the mixed acid fermentative pathway evolves hydrogen by formate hydrogen lyase

4 (FHL) during formate decomposition (Das, 2001). Second, in the NADH pathway, hydrogen

5 evolution occurs by hydrogenase via NADH oxidation (Tanisho et al., 1998). After irradiation of E.

6 aerogenes by 60Co-γ-rays, the formate or NADH pathway was intensified to promote hydrogen

7 production.

8 More than ten generations of E. aerogenes ZJU1 were cultivated to evaluate the hereditary

9 stability. 1 mL bacterial cell (first-generation) culture was respectively inoculated into three bottles

10 of 100 mL LB medium. Batch cultivations were performed aerobically in a shaker incubator at 37

11 °C and 180 rpm for 6–8 h until the cells reached the mid-log phase. Then, 6 mL of bacterial cell

12 culture was introduced in six tubes and preserved with record number (second-generation). The rest

13 culture of E. aerogenes ZJU1 (second-generation) was used for fermentation to assess the hydrogen

14 productivity. The third-generation of E. aerogenes ZJU1 would be obtained during next batch

15 cultivations of second-generation. The process would be repeated until fermentation of E.

16 aerogenes ZJU1 (tenth-generation) had been performed. As shown in Fig. 4, the hydrogen yield of

17 the mutant E. aerogenes ZJU1 was constant within ten subcultures. The hereditary stability of E.

18 aerogenes ZJU1 for hydrogen productivity was maintained after ten subcultures. Therefore, a stable

19 E. aerogenes mutant irradiated by 60Co γ-rays with high hydrogen production was obtained.

20 3.4. Metabolic by-products

12
1 The metabolic by-products of both strains were measured every 12 h during hydrogen

2 fermentation. The by-products were mainly composed of acetate, butyrate and ethanol for both

3 strains. As shown in Fig. 5, changes in the yields of these liquid by-products were analysed during

4 H2 fermentation. A comparison of the metabolites of both strains revealed that the final

5 concentrations of acetate and butyrate were increased from the wild strain to the mutant strain,

6 whereas that of the ethanol decreased. Final concentrations (mM) of acetate, butyrate and ethanol in

7 the mutant strain were 23.63 ± 0.57, 28.91 ± 0.16 and 17.68 ± 0.93, respectively, whereas those in

8 the wild strain were 19.84 ± 0.46, 12.01 ± 0.0032 and 32.08 ± 0.36, respectively. These changes in

9 metabolites corresponded to the enhanced hydrogen productivity in the mutant strain. According to

10 these metabolic pathways, the glucose substrate is degraded to pyruvate for ATP production, while

11 NAD+ is reduced to NADH (Hallenbeck and Ghosh, 2012). Pyruvate is converted into acetyl

12 coenzyme A and ferredoxin or formate, which could produce H2 (Cai et al., 2011). NADH is

13 released as reducing equivalents during glycolysis. For continued glycolysis, NADH needs to be

14 re-oxidised by producing H2, ethanol and organic acids. Competition was observed among H2,

15 ethanol and butyrate production because these reactions consume 1, 2 and 2 mol/mol of NADH,

16 respectively. However, acetate production does not require NADH (Cheng et al., 2013). Two

17 ethanol units or one butyrate unit can be produced from one glucose unit. Two ethanol units can

18 consume four NADH units, whereas one butyrate unit only needs two NADH units. The wild strain

19 produced less H2 because metabolism favours ethanol production to maintain the balance of

20 reducing equivalents (Zhang et al., 2009). For the mutant strain, more NADH was used to produce

21 H2 but less NADH was consumed to produce the by-products. More acetate and butyrate were

13
1 produced compared with ethanol; thus, less NADH was required. The metabolism of E. aerogenes

2 ZJU1 has changed after irradiation of 60Co γ-rays. These changes in metabolites correspond to the

3 enhanced hydrogen productivity in the mutant strain.

4 4. Conclusion

5 This study demonstrated that irradiation with 60Co γ-rays can alter the hydrogenase activity

6 and metabolism of E. aerogenes. The E. aerogenes ZJU1 mutant with larger yellow circles was

7 screened. The hydrogen yield of E. aerogenes ZJU1 was 2.41 mol/mol glucose, which was 81.8%

8 higher than the wild strain. The hereditary stability of E. aerogenes ZJU1 for hydrogen productivity

9 was maintained after ten subcultures. Genetic analyses will contribute to figuring out the key genes

10 of E.aerogenes in hydrogen fermentation in future. It is promising to utilize the mutated E.

11 aerogenes to facilitate fermentative hydrogen production from biomass waste in industrial

12 applications.

13 Acknowledgements

14 This study was supported by the National Key Technology R&D Program-China

15 (2015BAD21B01), National Natural Science Foundation-China (51476141), Zhejiang Provincial

16 Natural Science Foundation-China (LR14E060002), Shandong Provincial Natural Science

17 Foundation-China (ZR2014CL001).

18

19 References

20 [1] Angenent, L.T., K.arim, K., Al-Dahhan, M.H., Wrenn, B.A., Domíguez-Espinosa, R., 2004. Production of

21 bioenergy and biochemicals from industrial and agricultural wastewater. Trends Biotechnol. 22, 477–485.
14
1 [2] Cai, G., Jin, B., Monis, P., Saint, C., 2011. Metabolic flux network and analysis of fermentative hydrogen

2 production. Biotechnol. Adv. 29, 375–387.

3 [3] Carrieri, D., Ananyev, G., Dismukes, G.C., 2008. Renewable hydrogen production by cyanobacteria: Nickel

4 requirements for optimal hydrogenase activity. Int. J. Hydrogen Energy 33, 2014–2022.

5 [4] Cheng, H.H., Whang, L.M., Lin, C.A., Liu, I.C., Wu, C.W., 2013. Metabolic flux network analysis of

6 fermentative hydrogen production: Using Clostridium tyrobutyricum as an example. Bioresour. Technol. 141,

7 233–239.

8 [5] Cheng, J., Feng, J., Sun, J., Huang, Y., Zhou, J., Cen, K., 2014. Enhancing the lipid content of the diatom

9 Nitzschia sp. by 60 Co-γ irradiation mutation and high-salinity domestication. Energy 78, 9–15.

10 [6] Converti, A., Perego, P., 2002. Use of carbon and energy balances in the study of the anaerobic metabolism

11 of Enterobacter aerogenes at variable starting glucose concentrations. Appl. Microbiol. Biotechnol. 59,

12 303–309.

13 [7] Das, D., Veziroǧlu, T.N., 2001. Hydrogen production by biological processes: a survey of literature. Int. J.

14 Hydrogen Energy 26, 13–28.

15 [8] Feng, J., Cheng, J., Cheng, R., Zhang, C., Zhou, J., Cen, K., 2015. Screening the diatom Nitzschia sp.

16 re-mutated by 137Cs-γ irradiation and optimizing growth conditions to increase lipid productivity. J. Appl.

17 Phycol. 27, 661–672.

18 [9] Hallenbeck, P.C., Ghosh, D., 2012. Improvements in fermentative biological hydrogen production through

19 metabolic engineering. J. Environ. Manage. 95 Suppl, S360-S364

15
1 [10] Hawkes, F.R., Hussy, I., Kyazze, G., Dinsdale, R., Hawkes, D.L., 2007. Continuous dark fermentative

2 hydrogen production by mesophilic microflora: Principles and progress. Int. J. Hydrogen Energy 32,

3 172–184.

4 [11] Holladay, J.D., Hu, J., King, D.L., Wang, Y., 2009. An overview of hydrogen production technologies.

5 Catal. Today 139, 244-260.

6 [12] Jayasinghearachchi, H.S., Sarma, P.M., Singh, S., Aginihotri, A., Mandal, A.K., Lal, B., 2009. Fermentative

7 hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea

8 buried crude oil pipelines. Int. J. Hydrogen Energy 34, 7197–7207.

9 [13] Jones, P.R., 2008. Improving fermentative biomass-derived H2-production by engineering microbial

10 metabolism. Int. J. Hydrogen Energy 33, 5122–5130.

11 [14] Kothari, R., Buddhi, D., Sawhney, R.L., 2008. Comparison of environmental and economic aspects of

12 various hydrogen production methods. Renew. Sustain. Energy Rev. 12, 553–563.

13 [15] Kovacs, E., Keresztes, A., 2002. Effect of gamma and UV-B/C radiation on plant cells. Micron 33,

14 199–210.

15 [16] Lin, C., Chang, C., Hung, C., 2008. Fermentative hydrogen production from starch using natural mixed

16 cultures. Int. J. Hydrogen Energy 33, 2445–2453.

17 [17] Lin, R., Cheng, J., Ding, L., Song, W., Zhou, J., Cen, K., 2015. Inhibitory effects of furan derivatives and

18 phenolic compounds on dark hydrogen fermentation. Bioresour. Technol. 196, 250–255.

19 [18] Lu, W., Wen, J., Jia, X., Sun, B., Chen, Y., Liu, M., 2008. Effect of He-Ne laser irradiation on hydrogen

20 production by Enterobacter aerogenes. Int. J. Hydrogen Energy 33, 34–42.

16
1 [19] Lu, Y., Wang, L., Ma, K., Li, G., Zhang, C., Zhao, H., Lai, Q., Li, H.P., Xing, X.H., 2011. Characteristics of

2 hydrogen production of an Enterobacter aerogenes mutant generated by a new atmospheric and room

3 temperature plasma (ARTP). Biochem. Eng. J. 55, 17–22.

4 [20] Nitsch, J., Klaiss, H., Meyer, J., 1992. The contribution of hydrogen in the development of renewable

5 energy sources. Int. J. Hydrogen Energy 17, 651–663.

6 [21] Peck, H.D., Gest, H., 1956. A new procedure for assay of bacterial hydrogenases. J. Bacteriol. 71, 70–80.

7 [22] Rachman, M.A., Furutani, Y., Nakashimada, Y., Kakizono, T., Nishio, N., 1997. Enhanced hydrogen

8 production in altered mixed acid fermentation of glucose by Enterobacter aerogenes. J. Ferment. Bioeng. 83,

9 358–363.

10 [23] Ren, N.Q., Zheng, G.X., Li, Y.F., Lin, H.L., Li, J.Z., 2006. Mutagenesis and selection of high efficiency

11 hydrogen-producing mutants by ultraviolet radiation. J. Harbin Inst. Technol. 13, 635–639.

12 [24] Shi, Q.Q., Wu, S.G., 2013. Industrial microbe breeding, fourth ed. Science Press, Beijing.

13 [25] Song, W., Cheng, J., Zhao, J., Carrieri, D., Zhang, C., Zhou, J., Cen, K., 2011. Improvement of hydrogen

14 production by over-expression of a hydrogen-promoting protein gene in Enterobacter cloacae. Int. J.

15 Hydrogen Energy 36, 6609–6615.

16 [26] Su, H., Cheng, J., Zhou, J., Song, W., Cen, K., 2009. Improving hydrogen production from cassava starch by

17 combination of dark and photo fermentation. Int. J. Hydrogen Energy 34, 1780–1786.

18 [27] Tanisho, S., 1999. Hydrogen production by facultative anaerobe Enterobacter aerogenes. BioHydrogen 6,

19 273–279.

17
1 [28] Tanisho, S., Kamiya, N., Wakao, N., 1989. Hydrogen evolution of Enterobacter aerogenes depending on

2 culture pH: mechanism of hydrogen evolution from NADH by means of membrane-bound hydrogenase.

3 Biochim. Biophys. Acta 973, 1–6.

4 [29] Tanisho, S., Kuromoto, M., Kadokura, N., 1998. Effect of CO2 removal on hydrogen production by

5 fermentation. Int. J. Hydrogen Energy 23, 559–563.

6 [30] Yokoi, H., Ohkawara, T., Hirose, J., Hayashi, S., Takasaki, Y., 1995. Characteristics of hydrogen production

7 by aciduric Enterobacter aerogenes strain HO-39. J. Ferment. Bioeng. 80, 571–574.

8 [31] Zhang, C., Lv, F.X., Xing, X.H., 2011. Bioengineering of the Enterobacter aerogenes strain for biohydrogen

9 production. Bioresour. Technol. 102, 8344–8349.

10 [32] Zhang, C., Ma, K., Xing, X.H., 2009. Regulation of hydrogen production by Enterobacter aerogenes by

11 external NADH and NAD+. Int. J. Hydrogen Energy 34, 1226–1232.

12 [33] Zorin, N.A., 1986. Redox properties and active center of phototrophic bacteria hydrogenases. Biochimie 68,

13 97–101.

14

18
1 List of Figures and Tables:

2 Fig.1 Lethality percentage of hydrogen-producing bacteria E. aerogenes irradiated by different

3 doses of 60Co γ-rays.

4 Fig.2 Enhanced hydrogenase activity of efficient E. aerogenes mutant irradiated by 60Co γ-rays and

5 screened with color-circle method.

6 Fig.3 Hydrogen production rate (a) and cumulative hydrogen yield (b) of efficient E. aerogenes

7 mutant irradiated by 60Co γ-rays and screened with color-circle method.

8 Fig.4 Hereditary stability of efficient E. aerogenes mutant irradiated by 60Co γ-rays for hydrogen

9 productivity after 10 subcultures

10 Fig.5 Metabolism by-products of efficient E. aerogenes mutant irradiated by 60Co γ-rays and

11 screened with color-circle method during hydrogen fermentation.

12 Table 1 Dynamic parameters of fermentative hydrogen production of efficient E. aerogenes mutant

13 irradiated by 60Co γ-rays and screened with color-circle method

14
15

19
1

100

80
Lethality percentage (%)

60

40

20

0
0 200 400 600 800 1000
Nuclear irradiation dose (Gy)
2

3 Fig. 1 Lethality percentage of hydrogen-producing bacteria E. aerogenes irradiated by different

4 doses of 60Co γ-rays.

5

20
1

200
E. aerogenes ZJU1 (mutant)

Hydrogen production (ml / g-DW)

160 157.4±8.9 ml-H2/ (g-DW·h)

E. aerogenes (wild)
120 E. aerogenes ZJU1 (mutant)

80

40
E. aerogenes (wild)
89.8±7.5 ml-H2/ (g-DW·h)
0
0.0 0.2 0.4 0.6 0.8 1.0
Experimental time (h)
2

3 Fig.2 Enhanced hydrogenase activity of efficient E. aerogenes mutant irradiated by 60Co γ-rays and

4 screened with color-circle method.

21
 a
30
E.aerogenes

Hydrogen production rate (ml/(L•h))

(wild)
25 E.aerogenes ZJU1
(mutant)

20

15

10

0
0 12 24 36 48 60 72 84 96 108
Fermentative time (h)
1

b 350
E.aerogenes (wild)
E.aerogenes ZJU1 (mutant)
Cumulative hydrogen yield (ml/g)

300

250

200

150

100

50

0
0 12 24 36 48 60 72 84 96 108
Fermentative time (h)
2

3 Fig.3 Hydrogen production rate (a) and cumulative hydrogen yield (b) of efficient E. aerogenes

4 mutant irradiated by 60Co γ-rays and screened with color-circle method.

5
6
7

22
1

320

280

Cumulative hydrogen yield (ml/g)

240

200

160

120

80

40

0
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
Subculture
2
3 Fig.4 Hereditary stability of efficient E. aerogenes mutant irradiated by 60Co γ-rays for hydrogen
4 productivity after 10 subcultures
5

23
1

25 Acetate-E.aerogenes (wild)
20 Acetate-E.aerogenes ZJU1 (mutant)

15

Concentrations of metabolites (mM)

10
5
0
30 Butytate-E.aerogenes (wild)
Butytate-E.aerogenes ZJU1 (mutant)
20

10

0
30 Ethanol-E.aerogenes (wild)
Ethanol-E.aerogenes ZJU1 (mutant)
20

10

0
0 12 24 36 48 60 72 84 96 108

Fermentative time (h)

2
3

4 Fig.5 Metabolism by-products of efficient E. aerogenes mutant irradiated by 60Co γ-rays and

5 screened with color-circle method during hydrogen fermentation.

24
1

2 Table 1. Dynamic parameters of fermentative hydrogen production of efficient E. aerogenes mutant

3 irradiated by 60Co γ-rays and screened with color-circle method

Kinetic model parameters

Bacterial strains H2 yield Roverall
Hm Rm λ R2
(ml/g) (ml/l/h) (ml/g) (ml/g/h) (h)

E.aerogenes
165.6 9.2 168.3 5.0 27.2 0.9814
(wild)
E.aerogenes ZJU1
301.0 15.8 311.4 9.4 32.7 0.9918
(mutant)

25
 60
1 >Enterobacter aerogenes cells were mutated by nuclear irradiation of Co γ-rays

2 >E. aerogenes mutants were screened with larger colour circles of acid by-products

3 >Hydrogenase activity of the E. aerogenes ZJU1 mutant improved by 75.3%

4 >Hydrogen yield (301 mL H2/g glucose) with mutant was higher by 81.8% than the wild

26